含弓形虫ROP1基因真核表达重组质粒DNA免疫小鼠的研究Ⅴ.IFN-γ基因真核表达重组质粒的构建及其在DNA免疫中基因佐剂的作用

VACCINATING MICE WITH EUKARYOTIC EXPRESSION RECOMBINANT PLASMID DNA CODING RHOPTRY PROTEIN 1 GENE FROM TOXOPLASMA GONDII V.Constructing Eukaryotic Expression Recombinant Plasmid Containing Interferon Gamma Gene and Its Effects as a Genetic A

目的 构建ＩＦＮ－γ基因真核表达重组质粒作为基因佐剂，观察其与ｐｃＤＮＡ３－ＲＯＰ１（ｐｃ－ＲＯＰ１） ＤＮＡ共同免疫小鼠所诱导的免疫应答。方法 将ＩＦＮ－γ基因片段定向插入真核表达载体ｐｃＤＮＡ３，双酶切鉴定，获得ｐｃＩＦＮ 重组子；碱裂解法大量制备，经肌肉注射免疫ＢＡＬＢ／ｃ小鼠，每只鼠注射ｐｃＩＦＮ、ｐｃ－ＲＯＰ１ 各１００μｇ，两周后同量加强免疫一次，以ｐｃＤＮＡ３空质粒及生理盐水组为对照。分别于免疫后第３０ 天、５０ 天、７０天共三次用ＭＴＴ法测定小鼠脾脏Ｔ淋巴细胞增殖活性及ＮＫ细胞活性；双夹心ＥＬＩＳＡ 测定血清细胞因子ＩＦＮ－γ、ＩＬ－２及酶法测定ＮＯ含量；ＥＬＩＳＡ法测定ＩｇＧ抗体滴度。结果 构建成功的作用下，该两项指标均明显提高，且ＩＦＮ－γ、ＩＬ－２ 及ＮＯ水平均较不加佐剂组显著提高（Ｐ＜ ０．０１）；而对ＩｇＧ抗体滴度无显著影响（Ｐ＞ ０．０５。结论 ＩＦＮ－γ基因佐剂具有协同ｐｃ－ＲＯＰ１ＤＮＡ免疫的作用，可增强免疫鼠细胞免疫应答，ＩＦＮ－γ。

Aim Constructing the eukaryotic expresion recombinant plasmid pcIFN γ as a enetic adjuvant,to observe the immuno responses which was elicited by pcDNA3 ROP1(pc ROP1)with pcIFN γ DNA vaccine in mice.Methods\ Directional inserting the fragment of IFN γ gene into eukaryotic expression plasmid pcDNA3,the recombinant plasmid was identified by digesting with two restrictive enzymes.Largescale preparation of plasmid DNA by alkaline lysis,the DNA were injected through muscles of left leg in each mouse at the dosage of 100μg pcIFN and pcROP1 respectively.A booster vaccine was given at the same dosage after two weeks.Control groups were infected with pcDNA3 blank plasmid and normal saline respectively.After 30,50 and 70 days of the booster injection,following tests were carried out 3 times separately:the proliferation activity of T lymph cells and the activity of NK cells by means of MTT assay,Cytokines of IFN γ,IL 2 by sandwich ELISA and the titer of IgG antibody by ELISA.Results\ The recombinant plasmid,pcIFN,was constructed.the activity proliferation of spleen T lymph cells and NKC killing activity,IFN γ,IL 2 and NO in the pc ROP1 with pcIFN experiment group were higher than that of in the only pc ROP1 groups.However,the titer of IgG antibody in former group did not increase evidently compared with that of the control ones.Conclusion\ The genetic adjuvant,pcIFN,could work in coordination with pcROP1 enhancing the cellular immuno rseponse in DNA vaccinated mice.